Controlling anammox speciation and biofilm attachment strategy using N-biotransformation intermediates and organic carbon levels

Conventional nitrogen removal in wastewater treatment requires a high oxygen and energy input. Anaerobic ammonium oxidation (anammox), the single-step conversion of ammonium and nitrite to nitrogen gas, is a more energy and cost effective alternative applied extensively to sidestream wastewater treatment. It would also be a mainstream treatment option if species diversity and physiology were better understood. Anammox bacteria were enriched up to 80%, 90% and 50% relative abundance, from a single inoculum, under standard enrichment conditions with either stepwise-nitrite and ammonia concentration increases (R1), nitric oxide supplementation (R2), or complex organic carbon from mainstream wastewater (R3), respectively. Candidatus Brocadia caroliniensis predominated in all reactors, but a shift towards Ca. Brocadia sinica occurred at ammonium and nitrite concentrations > 270 mg NH4–N L−1 and 340 mg NO2–N L−1 respectively. With NO present, heterotrophic growth was inhibited, and Ca. Jettenia coexisted with Ca. B. caroliniensis before diminishing as nitrite increased to 160 mg NO2–N L−1. Organic carbon supplementation led to the emergence of heterotrophic communities that coevolved with Ca. B. caroliniensis. Ca. B. caroliniensis and Ca. Jettenia preferentially formed biofilms on surfaces, whereas Ca. Brocadia sinica formed granules in suspension. Our results indicate that multiple anammox bacteria species co-exist and occupy sub-niches in anammox reactors, and that the dominant population can be reversibly shifted by, for example, changing nitrogen load (i.e. high nitrite concentration favors Ca. Brocadia caroliniensis). Speciation has implications for wastewater process design, where the optimum cell immobilization strategy (i.e. carriers vs granules) depends on which species dominates.


Results
Start-up period reduced and N removal activity enhanced with NO supplementation. Anammox bacteria were successfully enriched under all tested enrichment conditions albeit with varying start-up times. Start-up period was the shortest in R2 supplemented with NO and anammox activity was observed within 20 days of inoculation compared to 39 days for R1, operated under standard enrichment conditions (Figs. 1A,B). In the presence of complex organic carbon in R3, anammox activity was only detected after 50 days of operation (Fig. 1C). In both R1 and R2, ammonium and nitrite concentrations were increased to 280 mg N L −1 and 350 mg N L −1 , respectively (Fig. 1A,B), above which anammox activity was inhibited. In addition to the shorter start-up period, a shorter hydraulic retention time (HRT) was applied to R2 than R1 due to higher N removal rates of 1200 mg N L −1 day −1 compared to 800 mg N L −1 day −1 under stable operation (Fig. 1A,B). Despite the higher loading rate, the suspended solid concentrations were comparable in both reactors indicating a higher specific N removal activity for R2 than R1. A significantly lower N loading rate of 121 ± 6 mg N L −1 day −1 was achieved in R3. Final effluent ammonium concentration dropped steadily from day 58 to 80, with the reactor displaying stable ammonium removal activity by anammox bacteria henceforth. Residual nitrite in the effluent also decreased gradually from day 60 to 100 along with a decrease in ammonium concentration (Fig. 1C). The Either high N load or concentration, or both, could shift the dominant anammox bacteria species from Ca. B. caroliniensis to Ca. B. sinica. The microbial communities of three reactors were differentiated by day of operation (R = 0.53, p = 0.007) and by the use of different reactors, i.e. R1 vs R2 vs R3 (R = 0.38, p = 0.001). In addition, the R1 and R2 communities at different levels of N showed relatively strong dissimilarity (R = 0.48 and 0.57, respectively, with p = 0.001 for both). Along with the increase in anammox activity, a shift in the functional anammox bacteria was observed in R1 and R2 along with increasing N load, but not in R3 where low N load was maintained. 16S rRNA gene amplicon sequencing showed that anammox bacteria were below the detection limit (< 0.018%) at the start of reactor operation for all three reactors. In R1 and R2, opera- caroliniensis ( ) and Ca. B. sinica ( ) and correlated non-anammox bacteria OTUs affiliated to Anaerolineaceae ( ) and Fimbriimonadia ( ) are shown in the lower panels of (A,B); Ca. Jettenia ( ) was also detected in R2 (B). Ca. B. caroliniensis, the only dominant anammox bacteria in R3, is shown in (C) along with correlated non-anammox bacteria OTUs affiliated to Comamonadaceae ( ) and Ca. Aquirestis ( ) dominating at a different stage. The relative abundance of total anammox bacteria is highlighted as area plot ( ) in each graph. Red dotted lines denote the time points at which anammox biofilm was scraped from the wall of the reactor into suspension. The detailed chemical and microbial community (with OTUs > 5% at any analysed time point) can be found in Supplementary Fig. S1 www.nature.com/scientificreports/ tional taxonomic units (OTUs) annotated to anammox bacteria increased progressively to 80% (day 110) and 90% (day 95) of the relative abundances of OTUs, respectively, at influent nitrite concentration > 200 mg N L -1 (Fig. 1A,B). The microbial communities of R1 and R2 at different levels of N level showed relative strong dissimilarity (R = 0.48 and 0.57, respectively, with p = 0.001 for both). The microbial community at high N load period, on the other hand, was not highly differentiated (R = 0.29, p = 0.003). Despite the increase in relative abundance of multiple OTUs affiliated to anammox bacteria in both R1 and R2, a single OTU annotated to Ca. Brocadia, identified as Ca. B. caroliniensis by clone library analysis (Fig. 2), dominated throughout the first 120 days of reactor operation. Ca. B. caroliniensis increased during enrichment to 50% relative abundance in R1 and R2. However, a further increase in influent ammonium and nitrite concentrations beyond 220 mg N L −1 from day 100 (N loading rate of 500 mg N L −1 -day −1 for R1 and 750 mg N L −1 -day −1 for R2) resulted in the gradual increase of Ca. Brocadia_2, identified as Ca. B. sinica by clone library analysis (Fig. 2). A decrease in Ca. B. caroliniensis was also observed (Fig. 1A,B). Beyond 180 days of reactor operation, Ca. B. sinica increased to 33% and 42% in relative abundance in R1 and R2, respectively, while Ca. B. caroliniensis decreased to less than 10% in relative abundance in both reactors. In the presence of organic carbon, Ca. B. caroliniensis was the most dominant anammox taxon throughout the enrichment process in R3 operated at a low N loading rate of 120 mg N L −1 day −1 (Figs. 1C). However, the relative abundance of total anammox bacteria was significantly lower in R3 (~ 50%) than in R1 and R2 (~ 80%), suggesting a more competitive environment for anammox bacteria in the presence of organic carbon. Fluorescent in situ hybridization (FISH) analysis ( Fig. 3D-F) on R1 (day 80) and R2 (day 675) further indicated that Ca. B. sinica dominated in those reactors while Ca. B. caroliniensis remained as the only anammox bacteria detected in R3 (day 683). The designed species-specific FISH probes served to observe gradual population shifts during reactor operation in response to changes in the controlling factors.
While other OTUs affiliated to genus Ca. Brocadia were also detected, their relative abundance was less than 10% ( Supplementary Fig. S1). The presence of these Ca. Brocadia OTUs is likely due to different strains of Ca. Brocadia or sequencing errors as only low relative abundances were detected. Aside from Ca. Brocadia, OTUs affiliated to Ca. Jettenia emerged to coexist with Ca. B. caroliniensis only in R2 (with continuous supply of NO), suggesting that the presence of NO may provide a competitive advantage for Ca. Jettenia. However, Ca. Jettenia diminished with increasing N loading rate (i.e. after day 102).
Excess NO availability over nitrite selects for Ca. Jettenia. Ca. Jettenia was only observed in R2 but not in R1 and R3, which could be due to the presence of NO. However, during the enrichment process it was unclear whether the NO effect was due to imposition of an oxidative stress or because it was used as a substrate for ammonium oxidation. This could not be assessed because nitrite was in excess during the enrichment. Hence, to investigate whether NO is consumed, nitrite was systematically depleted in R2 (Phase II) while dosing the same amount of NO (Phase I). After 76 days of nitrite depletion, nitrite was gradually reintroduced in Phase III (Fig. 4). www.nature.com/scientificreports/ Reducing the nitrite concentration resulted in a decrease in the relative abundance of Ca. B. sinica in suspension coincident with an increase in NO consumption rate (NCR) while a slight increase was observed for both Ca. B. caroliniensis and Ca. Jettenia in Phase II (Fig. 4). The presence of Ca. B. caroliniensis, absent in Phase I, was also detected through FISH analysis in Phase II (Fig. 4). Batch activity tests conducted during Phase II also showed a decline in the nitrite-dependent ammonium removal rate from 1352 mg N g MLVSS −1 day −1 prior to nitrite depletion (Phase I) to 681 mg N g MLVSS −1 day −1 after nitrite depletion (Phase II). Nevertheless, the overall activity in R2 was still higher than that in R1 even with a specific nitrite-dependent ammonium removal rate of 575 mg N g MLVSS −1 day −1 (Fig. 5). Under normal operation (Phase I), the NO-dependent ammonium oxidation in the absence of nitrite in both the reactors was insignificant, further supporting the hypothesis that nitrite rather than NO is the preferred electron acceptor and ammonium removal cannot be achieved by Ca. B. sinica via direct coupling to NO reduction. In contrast, the ammonium oxidation rates with NO in the absence of nitrite increased more than five times in R2 at 440 mg N g MLVSS −1 day −1 after nitrite depletion in Phase II compared to 33 and 80 mg N g MLVSS −1 day −1 in R1 and R2, respectively in Phase I under normal operation (Fig. 5). This suggests the selection of anammox bacteria species capable of utilizing externally supplied NO to oxidize ammonium.
At the start of experimental Phase III, Ca. B. caroliniensis was found to be in higher relative abundance than Ca. B. sinica in biofilms forming on the wall of the reactor (Fig. 4). Ca. Jettenia also showed a recovery, albeit at low abundance (observed in wall samples) in the absence of nitrite (Fig. 4). While it cannot be confirmed whether this was a consequence of nitrite depletion in Phase II, the relative abundances of Ca. B. caroliniensis and Ca. Jettenia in the biofilm collected from wall, were higher than under normal operation (Phase I in Fig. 6). A clear reversal from Ca. B. caroliniensis to Ca. B. sinica was observed once nitrite was reintroduced between 650 and 680 days (Fig. 4). Similar to that detected in Phase II, the increase in relative abundance of Ca. B. sinica coincided with the recovery of nitrite-dependent ammonium oxidizing activity of 1163 mg N g MLVSS −1 day −1 which is comparable to that in Phase I (1352 mg N g MLVSS −1 day −1 ) ( Fig. 4). In addition, the NO-dependent ammonium oxidizing activity also decreased from 440 (Phase II) to 102 (Phase III) mg N g MLVSS −1 day −1 (Fig. 5), further suggesting that increased NO consumption is likely linked to the increased abundance of Ca. B. caroliniensis or Ca. Jettenia or both. An extended period under nitrite limitation could have further enhanced the recovery of Ca. Jettenia and Ca. B. caroliniensis to outcompete Ca. B. sinica. Nevertheless, this part of the study supports a link between nitrite and NO, species selection and their preferred growth mode.

Prevailing anammox bacteria taxa exhibit different biomass morphologies. The predominant
anammox bacteria also displayed a distinct preference for attached growth under the various enrichment conditions. Anammox bacteria biomass was present mainly as suspended granules in R1 and R2 while biofilms attached to the reactor surface dominated in R3 ( Supplementary Fig. S2). Following the transfer of biofilm from the wall of the reactor into suspension (as indicated by dotted line in Fig. 1A-C), a greater increase in the MLVSS of R3 (around 1.5 g L −1 at day 230) was observed compared to the other two reactors (less than 0.3 g L −1 at days www.nature.com/scientificreports/ 160, 209, and 258 in R1 and 153, 216, and 253 in R2), suggesting more attached biofilm growth in the primary effluent-fed reactor (R3, Fig. 1C). In addition, following the reintroduction of nitrite into R2 during experimental Phase III, Ca. B. sinica showed a downward trend, more evident in the biofilm samples collected from the walls of the reactor (Fig. 4) than in suspension (p = 0.038). The difference in relative abundance between wall and suspension samples indicates a preference of Ca. B. caroliniensis for attached growth. There was no significant difference between anammox bacteria populations of biomass samples collected from the wall and suspension in R1 and R2, with Ca. B. sinica as the dominant anammox bacteria. However, the relative abundance of predominant Ca. B. caroliniensis was four times higher in the biomass collected from the wall than in suspension for R3 (Fig. 6), further indicating that this species has a tendency towards attached growth (Fig. 6). While granules were found to form in R1 and R2 (Fig. 3A,B), with an average particle size of 1.52 and 1527.9 ± 0.078 μm (Supplementary Table S2) respectively with R = 0.35 (p = 0.006) between the two reactors, the morphology of aggregates in R3 was more floc-like (Fig. 3C)  Ca. Jettenia ( ) in suspended (highlighted as "Granules in suspension" on the x-axis) and attached growth biomass (highlighted as "biofilm on the wall" on the x-axis), and the NO consumption rate (NCR, ) of R2, fed with synthetic waste water with ammonium, nitrite and continuous supply of nitric oxide. Influent nitrite (NO 2, ) was adjusted from normal (Phase I), to depletion (Phase II) and repletion (Phase III). The relative abundance of total anammox bacteria is highlighted as area plot (  Fig. S3).   29,33 , which is the highest maximum growth rate ever reported for anammox bacteria. This indicates that Ca. B. sinica are r-strategists and would grow at high N loading rates and ammonium and nitrite concentrations 33 , as observed in our study. However, a similar estimation for Ca. B. caroliniensis is missing. Metagenomic analysis revealed that Ca. B. caroliniensis have multiple copies of nitrite/formate transporters (focA) that provide a competitive advantage at low nitrite concentrations due to a low intrinsic nitrite affinity constant 14 . This could potentially help them scavenge nitrite from heterotrophic denitrifiers, especially when the competition for nitrite is higher in the presence of organic carbon.
Despite the wide range of environmental conditions applied across the three enrichment reactors, Ca. Brocadia remained the most dominant phylotype throughout the enrichment process, while Ca. Kuenenia and Ca. Anammoxoglobus commonly found in engineered systems were not detected. Although abundances of all anammox bacteria were low in the inoculum, operational conditions, especially the relatively high nitrogen load, contributed to the enrichment of Ca. Brocadia over others 34 . NO can select for specific anammox bacteria taxa. NO was provided in R2 to exert oxidative stress and also to potentially select for NO-utilizing anammox bacteria 35 . While the presence of NO did not appear to suppress the growth of anammox bacteria, it may have provided a competitive advantage to Ca. Jettenia in R2, which increased to a maximum relative abundance of 23% along with Ca. B. caroliniensis at low N loading (Fig. 1). Little is known about the ecological and metabolic drivers of the niche of Ca. Jettenia, probably because they are generally less abundant than other genera of anammox bacteria 3 . Low nitrite concentrations were shown to encourage the proliferation of Ca. Jettenia over Ca. B. sinica 4 , consistent with this study. However, Ca. Jettenia was much less abundant than Ca. B. caroliniensis at low nitrite loading rates. Nevertheless, two phylogenetically distant anammox bacteria species, Ca. B. caroliniensis and Ca. Jettenia, were shown to coexist in the same system which supports the previous findings 3 . However, since microbial community monitoring is based on the 16S rRNA gene only, a bias may be induced when focusing on a single region of the gene. Further validation using a multiple primer sequencing approach could be undertaken to improve the accuracy of quantification 36 .
The coevolution of Ca. B. caroliniensis and Ca. Jettenia might also suggest that Ca. B. caroliniensis could utilize NO. A similar nitrite reduction pathway in Ca. B. caroliniensis and Ca. Jettenia was suggested after a nirK homologue was detected from the metagenomic analysis of Ca. B. caroliniensis 14,37 . The significant increase in NO-dependent ammonium oxidation following nitrite limitation was concomitant with the recovery of Ca. B. caroliniensis and Ca. Jettenia populations. A recent study reported the discovery of nirS in a Ca. Brocadia genome 38 , however only weak transcripts were found and this observation requires additional validation. It is conceivable that Ca. B. caroliniensis utilize a nirK homologue or a novel nitrite reductase employing the conventional NO-dependent pathway for hydrazine production or possess the ability to switch to a NO-dependent pathway in the absence of nitrite. An alternate pathway for NO production through oxidation of hydroxylamine by a hydroxylamine oxidoreductase (hao)-like protein was indeed detected by Park et al. 14 and Irisa et al. 39 . They proposed that this alternate pathway could potentially be activated under nitrite limitation 14 . NO was also shown to oxidize ammonium to dinitrogen gas under nitrite limitation in Ca. B. fulgida 21 and canonical nirS was absent in its genome 40 .
In contrast, NO-dependent ammonium oxidation was negligible at significantly higher NO concentrations, further corroborating the assertion that nitrite rather than NO was the preferred electron acceptor and that ammonium removal cannot be achieved by Ca. B. sinica via direct coupling to NO reduction (Fig. 5) www.nature.com/scientificreports/ et al. 42 revealed using 15 N-labeling experiments that ammonium was oxidized to dinitrogen via hydroxylamine as intermediate instead of NO in a Ca. Brocadia enrichment culture, with an electrode as electron acceptor. It is noted, however, that it is not possible to assign behaviours to species with absolute confidence in the absence of pure cultures. All anammox bacteria are non-cultivable, and the only option for attributing behaviours to species, and identifying their niches and optimum growth conditions, is phenomenological studies on enrichment reactors. Here, enrichments of between 50 and 80% were achieved, which is high for a population enrichment reactor 43 ; they rank among the highest anammox enrichments that have been achieved in an SBR so far 44 . An enrichment to 99.5% can be achieved by Percoll density centrifugation 45 , but the high biomass required hampers resolution of the microbial community at the species level. While membrane bioreactors have been used to enrich planktonic populations of Ca. B. sinica, Ca. Scalindua 29 and Ca. K. stuttgartsiensis 10 , our approach also enriched for species that prefer to grow in biofilms like B. caroliniensis and Ca. Jettenia.
The predominant anammox bacteria species determine biomass retention strategy. High retention of anammox bacteria in reactors is a crucial factor for optimal operation due to the slow growth rate of these bacteria. This can be achieved through biofilm attachment to carriers, formation of granular biomass aggregate and other separation techniques such as membrane filtration to prevent washout of anammox bacteria. The choice of biomass retention in anammox systems may be guided by the growth mode of prevailing anammox bacteria species and the coexisting microbial community under specific operational conditions. In this study, it was demonstrated that the anammox bacteria community and their aggregation states might be distinct under mainstream and sidestream conditions. Ca. B. caroliniensis, likely persisting under mainstream conditions, exhibited a preference for attached biofilm growth. High diversity and abundance of heterotrophic species in R3 was also observed in the presence of complex organic carbon. Particularly, Comamonadaceae remained one of the most abundant heterotrophs in the system both in suspension and in the biofilm. Comamonadaceae are commonly found in biofilm forming communities 46,47 suggesting their potential role in assisting biofilm formation. Attached growth of Ca. B. caroliniensis was also observed in a full-scale process treating anaerobic digester liquid supplied with glycerol as the external carbon source 14 . In this instance, carriers can be used to provide a large surface area to achieve high biomass retention 48 . Carriers supporting attached biofilm growth can be applied in various configurations, for instance, in rotating biological contactors 49 , moving bed biofilm reactors 50,51 and sequencing batch biofilm reactors 52 . However, Ca. B. sinica dominated anammox biofilms forming granules (as observed in R1 and R2 operated under side stream conditions), and can be separated physically using a hydrocyclone as with DEMON SBR systems 53 , lamella separators 54 or integrated fixed film activated sludge (IFAS) configurations with a settler 55 . A particular extracellular protein was found to be highly abundant in the extracellular matrix of the Ca. B. sinica granules which promotes biofilm formation across several length scales due to its ability to phase separate (droplets and gels) and promote adhesion 56 . This could explain the greater tendency of Ca. B. sinica to self-aggregate (i.e. in the absence of a substratum, unlike Ca. B caroliensis and Ca. Jettenia). Despite the absence of externally supplied organic carbon, heterotrophs belonging to the class Fimbriimonadia (phylum Armatimonadetes) and family Anaerolineaceae (phylum Chloroflexi) proliferated in R1 and R2 to a relative abundance of 10-15%. Gao et al. 57 suggested an important role of Anaerolineaceae as cores or carriers for granule formation in anammox sludge and their increase in abundance over time would suggest that they may have supported granulation in both R1 and R2. However, the role of heterotrophic bacteria and their interaction with anammox bacteria cannot be fully uncovered in this study and will require further investigation.

Conclusions
Multiple anammox bacterial species can be enriched from the same activated sludge. Ca. B. caroliniensis dominates at low N loads, both in the presence and absence of organic carbon and under nitrite limitation, and forms attached biofilms; Ca. B. sinica outcompetes it at higher N loads and forms granules; and NO supplementation promotes Ca Jettenia even though it still disappears at high nitrate concentrations. Thus, Ca. B. caroliensis likely dominates in mainstream wastewater anammox processes, where carriers would be the best biofilm retention strategy, Ca B. sinica in sidestream treatments with granules the best retention strategy, and Ca Jettenia is unlikely to be competitive in wastewater treatment systems. Collectively, this study provides insight into understanding the relationship of species selection, growth morphology and process conditions in mainstream and sidestream applications with important implications in process design, control and management of the anammox process at the species level in full scale waste water treatment systems. www.nature.com/scientificreports/ impose oxidative stress. The selected concentration of 400 ppmv was lower than the previously reported tolerance threshold of 600 ppmv for anammox bacteria 22 . R1 and R2 were operated in cycles of 12 h, each cycle comprised of 5 min of feeding, 108 min of anoxic cycle, 67 min of settling and decanting. Initial ammonium and nitrite concentrations were maintained at 20 mg N L −1 within the reactor for the first seven weeks resulting in a hydraulic retention time (HRT) of 24 h (two litres of synthetic waste water were fed into the reactor each cycle). Upon achievement of 100% ammonium removal, NH 4 + and NO 2 − concentrations in the feed were then increased in steps of 20 mg N L −1 . Influent NO 2 − : NH 4 + was maintained at a molar ratio of 1.3 close to theoretical stoichiometry 59 . HRT was gradually decreased from 24 to 16 h for R1 and from 24 to 12 h for R2, in accordance with the N removal capacity. Thus, R2 was operated at a higher N loading rate compared to R1 due to shorter cycle time concomitant with higher N removal rates in R2. The pH was not controlled in either R1 or R2 and varied between 7.2 and 7.8.

Materials and methods
R3 was operated in cycles of 8-12 h, each cycle comprised of 2 h feeding, 5-9 h anoxic phase (depending on the cycle length applied), and 1 h of settling and decanting. Before settling, the reactor was sparged with Argon/ CO 2 for 5 min to strip out the nitrogen gas produced during the anoxic phase to improve sludge settle-ability. In each feeding period, 2 L of primary effluent supplemented with nitrite was added, resulting in a HRT of 16-24 h. Nitrite was adjusted according to the ammonium concentration at a molar ratio of 2:1 and stored in a chiller at 4 °C to minimize degradation. The nutrient composition of primary effluent was measured after addition of nitrite with average values shown in Supplementary Table S1. Slow feeding of 2 h was applied to minimise oxygen introduction and temperature shock from primary effluent stored in the chiller. The pH of the reactor was not controlled and varied between 7.6 and 8.5 due to denitrification activity. For enrichment purpose, the SRT was not controlled in all three reactors whereby sludge loss only occurred through sampling for nutrient and solids analyses (SRT estimated to be > 20 days).
A heating jacket was connected to maintain the SBR at 35 ± 0.05 °C for R1 and R2 and 33 ± 1 °C for R3. Dissolved oxygen (DO) concentration and pH were continuously monitored using Mettler Toledo InPro6050 DO sensor and Mettler Toledo-InPro 3250i pH sensor, respectively. Samples were collected periodically at the end of the cycle and filtered immediately with 0.2 µm filters for nutrient analyses. Mixed liquor samples were collected in the middle of the anoxic phase for DNA extraction. To determine the microbial composition of the biofilm on the reactor surface, biomass samples from the wall was collected from three random locations after draining the reactor at day 289 for R1, day 265 for R2 and 266 for R3. Both collected suspended and biofilm samples were snap-frozen in liquid nitrogen and stored at − 80 °C until extraction. Biofilm on the surface of the reactor was periodically cleaned to determine the concentration of total mixed liquor suspended solids (MLSS) and the volatile fraction (MLVSS) and the proportion of biomass that was in suspension versus attached growth. Suspended biomass samples were also collected for particle size analysis, and light microscopy imaging after stable enrichment was attained.

NO depletion and repletion experiment.
To further validate the effect of NO as a selection pressure for anammox bacteria species selection, R2 was subjected to gradual nitrite depletion and repletion while maintaining availability of NO as an electron acceptor across three experimental phases after stable operation was attained: Phase I -normal operation prior to nitrite depletion, ammonium and nitrite concentration in the feed were 280 and 350 mg N L −1 , respectively with continuous supply of NO at 400 ppmv in the gas phase (before day 563); Phase II-nitrite limited operation (day 564-640) whereby nitrite was reduced stepwise from 50 to 0 mg NO 2 -N L −1 while ammonium and NO were maintained at 50 mg NH 4 -N L −1 and 400 ppmv, respectively; Phase III (day 640-687) nitrite was gradually reintroduced from 0 to 70 mg NO 2 -N L −1 with the aforementioned ammonium and NO concentrations in Phase II. Suspended biomass samples were collected twice a week from the mixed liquor throughout the experiment however biofilm attached to the wall were only collected from Phase III due to the limited amount of wall biomass. At each experimental phase, batch activity tests were conducted in triplicate with 80 mg NH 4 + -N L −1 , 100 mg NO 2 -N L −1 and/or 400 ppmv NO in gas phase under the following conditions with (i) ammonium and nitrite only, (ii) ammonium, nitrite and NO, and (iii) presence of ammonium and NO only. The anammox activity of R1 under normal operation with ammonium and nitrite supplied as substrate served as the control. In all batch activity tests, mixed liquor samples were collected every 30 min and filtered through 0.22 µm Milipore filters for nutrient analysis.
Chemical analysis. All samples collected for nutrient analysis were measured for ammonium, nitrite and nitrate. Ammonium was measured using Hach ® kits, nitrate and nitrite were analyzed using ion chromatography (Prominence, Shimadzu). MLSS and MLVSS were analyzed according to the standard methods 60 . NO was measured in the gas phase using an online chemiluminescence analyzer (Model: 42i, Thermoscientific). Particle size analysis was carried out using laser diffraction particle size analyzer (Model: SALD-MS30, Shimadzu), ANOSIM analysis were carried out on the particle size measurements on samples collected from different reactors.
Suspended biomass was collected from each reactor and subject to size analysis. 1 mL biomass was dispersed on the surface of petri dish, and images were taken by AxioObserver Z1 inverted epifluorescent microscope (Leica, Germany) with brick/seal function. Images were then analyzed with image J 61 Analyze particles function.